Liquid chromatography is used to separate the components of a sample substance by passing an eluent liquid containing the sample through a column. The components of the sample in the eluent stream have different retention times within the column, and therefore exit the column in a particular sequence depending upon the nature of the components of the sample and the nature of the column. The sequence of components is detected, for example, photometrically by measuring the intensity of light passing through the eluent stream. The intensity of light is converted into an electrical signal proportional to the absorbance of the light by the component through which the light is passing, and such signal can be plotted by a chart recorder to create a chromatogram for the sample substance.
A problem experienced in liquid chromatography is that components often have very similar retention times within the chromatograph column so that the peaks of the chromatogram representing such components are overlapping or convoluted. Overlapping of peaks on the chromatogram distorts the true location and shape of the peaks and makes it difficult to accurately measure the precise retention time of the component and the amplitude of its representative peak on the chromatogram. Overlapping also makes it difficult to determine when a single pure substance is passing through the detector. Such information is needed when the purity of a substance must be confirmed, or when chromatography is used to collect quantities of highly pure components.
Most attempts in the prior art to solve the problem of distinguishing between components having identical or nearly identical retention times have been directed to increasing the separation of the components by the chromatograph column. This approach can be successful if the overlapping is not too severe. However, in many cases it is not possible to obtain sufficient separation of the peaks representing the components.
By convention, the absorbance of a substance (Aw) is defined as: EQU Aw=a.times.b.times.c
where a is the absorbance constant of the substance at the wavelength w being examined, b is the length of the flow cell through which the eluent and the detected light are being passed, and c is the concentration of the absorbing substance. For two different wavelengths of light (w' and w") passing through the same flow cell, the ratio of the absorbance of the substance at the two wavelengths is a constant: EQU Aw'/Aw"=(a'.times.b.times.c)/(a".times.b.times.c)=constant.
This constant, Aw'/Aw" is known as the absorbance ratio, and can be used to characterize pure chemicals in the same manner as density, boiling point, melting point, refractive index or chromatographic retention time. However, if the measured absorbance at the two wavelengths, Aw' and Aw" represent retention times when more than one substance is present in the flow cell, the absorbance ratio is not a constant, but changes as the relative concentrations of the components within the flow cell change.
Absorbance ratio has formerly been measured by a sequential process of trapping the compound in the flow cell by stopping the flow, determining the absorbance at two wavelengths, and dividing the two absorbances to obtain the ratio. This method is slow, labor intensive and gives information at only one point.
Another known method for obtaining absorbance ratio is by using two separate detectors set at different wavelengths. The chromatograms obtained can be stored digitally in a computer and the two signals can then be divided to obtain the desired ratio. This method requires sophisticated data processing techniques, and cannot be done in real time because the two detector signals do not represent the same point in the chromatogram. Also, since the first flow cell will have a peak-spreading effect on the chromatogram, the second flow cell will not see the same concentration profile as the first and the peak ratio calculation will be in error.
A third method consists of running two chromatograms either simultaneously using two chromatographs or consecutively with a single device, with the detector(s) adjusted to detect at different wavelengths. The results are then compared either manually or by computer, resulting in the ratio, difference and/or sum. This method is time-consuming and inaccurate because the results depend on injecting exactly the same quantities of sample in the two runs. In practice, this is difficult to do with high accuracy without extraordinary precautions being taken.